Enamel is a mineralized tissue formed by the epithelially derived ameloblasts. The reciprocal interactions between epithelial cells of the enamel organ and dental mesenchymal cells lead to the differentiation of ameloblasts, which govern the synthesis of the enamel matrix. Studies of ameloblast function and differentiation have been limited by difficulties in isolation and maintenance of the cells in primary culture, as well as our limited knowledge of ameloblast biology. The goal of these proposed studies is to characterize ameloblast lineage cells in culture and to study their differentiation. We hypothesize that factors present in the local microenvironment of the ameloblasts will direct their differentiation and propose the following specific aims. Specific Aim1: To isolate, expand and maintain primary undifferentiated ameloblast lineage cells from single-cell colonies in vitro. Specific aim 2: To induce differentiation of ameloblast lineage cells to mature ameloblasts. Cells from the enamel organ epithelium will be cloned from single cells, characterized by the epithelial marker cytokeratin 14, and markers of preameloblasts including dentin sialoprotein (DSP) and amelogenin. Ameloblast differentiation will be induced by cell/cell interactions with dental mesencymal cells, extracellular matrix proteins, growth factors and calcium. The early-differentiated secretory ameloblasts will be characterized by upregulation of amelogenin, ameloblastin, and MMP-20. Mature ameloblasts will be characterized by upregulation of kallikrein 4 (KLK4), amelotin and Ae2. The identification of factors that drive ameloblast differentiation will allow us to better understand normal and pathological ameloblast biology, and how these cells engineer the formation of the enamel matrix. The regeneration of tooth enamel requires an understanding of the factors that control expansion and differentiation of ameloblast (enamel forming) lineage cells. In this proposal we propose to identify key factors that direct ameloblast lineage cell fate decisions as they form an enamel matrix. [unreadable] [unreadable] [unreadable]